1. Field of the Invention
The present invention relates to the use of homeobox peptides or peptides derived therefrom for the production of medicinal products.
2. Discussion of the Background
The name homeobox peptides denotes a family of related peptide sequences which occur in various animal species in the products of genes involved in embryogenesis.
Indeed, genes are known which are expressed at various stages of embryo development and whose expression controls the cell migration and differentiation phenomena involved in the morphogenesis of the organism.
These genes are called homeotic genes and their translational products are called homeoproteins.
One of these genes, which has been most particularly studied, is the Antennapedia gene of Drosophila; the analysis of this gene has made it possible to identify a DNA sequence of about 180 bp, called homeobox sequence.
This homeobox sequence has the characteristic of being highly conserved in many homeotic genes, and this not only in Drosophila, but also during the course of evolution, in various animal species. Homeobox sequences homologous to that of Drosophila have thus been found in all vertebrates including mammals ACAMPORA et al., NUCLEIC ACID RES., 17, 10385, (1989)!.
The homeobox sequence encodes a polypeptide sequence of 60 amino acids which corresponds to a structurally and functionally conserved region which is present in all homeoproteins, the homeodomain. The sequence of the homeodomain which is encoded by the homeobox sequence of the Antennapedia gene (SEQ ID NO: 1)is indicated below by way of example.
NH2-Arg Lys Arg Gly Arg Gln Thr Tyr Thr Arg Tyr Gln Thr Leu Glu Leu Glu Lys Glu Phe His Phe Asn Arg Tyr Leu Thr Arg Arg Arg Arg lle Glu lle Ala His Ala Leu Cys Leu Thr Glu Arg Gln lle Lys lle Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys Glu Asn-COOH.
The role and the mechanism of action of the homeodomain sequence have been the subject of various research studies. It is thus currently known that this sequence allows the binding of the homeoproteins to DNA in the region of consensus sequences containing the unit ATTA, which are present in the promoters or in the enhancer sequences of various genes, including the homeobox-containing genes themselves.
MULLER et al EMBO J., 7, 4299, (1988)! have cloned the homeobox sequence of Antennapedia and purified the corresponding polypeptide or homeobox peptide (pAntp). In the presence of a reducing agent, they obtained the polypeptide in the form of a monomer with a sedimentation coefficient of about 1 S and with an apparent molecular weight of 9040 Da (theoretical molecular weight, based on the peptide sequence=8545 Da).
In the absence of a reducing agent, the polypeptide preparation contained a high proportion of dimers corresponding to homeodomains attached to each other via disulphide bridges.
These same authors also showed that the polypeptide purified in monomeric form became bound to the DNA in the region of a sequence ANNNNCATTA, (SEQ ID NO: 2) which therefore contains the consensus sequence ATTA.
Other research work OTTING et al., EMBO J., 7, 4305, (1988)! has established that the homeodomain has a special structure (helix/.beta.-turn/helix) which is reported to be involved in the binding to DNA.
The homeobox peptide/DNA binding is reported to be the resultant:
of a high-affinity binding (K.sub.D 10.sup.-9 -10.sup.-10 M), involving the consensus sequence ATTA, PA1 and of a low-affinity binding (K.sub.D 10.sup.-6 -10.sup.-7 M), involving the wide groove of the DNA double helix.
A publication by KISSINGER et al. Cell, 63, 579-590, (1990)! describes a crystallographic study on an "engrailed homeodomain/DNA" complex. This study shows that the C-terminal portion of the homeodomain, comprising in particular the structure called helix 3 (amino acids 42-58 of the engrailed homeodomain), binds to the wide groove of the DNA. This binding is essentially brought about by hydrophilic interactions; this binding is reported to be independent of the presence of the consensus sequence.
All known homeodomains share the same helix/.beta.-turn/helix structure, despite some differences in their primary sequences.
Given the very high degree of conservation of the homeobox sequences from one species to another, it is considered that the properties of the homeobox peptide pAntp can be extended to other homeobox peptides which may differ in their sequence by a few amino acids but possess virtually identical functional properties. In the following text, the term "homeobox peptide" will designate any peptide having the full-length sequence of an homeodomain as defined above, and able to fold into the helix/.beta.-turn/helix structure. This includes, for instance, the peptide pAntp or any other member of the same family or of a related family, for example the "engrailed" family.
"Helix 3" is understood to mean the portion of an homeobox peptide as defined above, which is involved in the low-affinity binding with the wide groove of the DNA. According to the definition provided by BURGLIN Cell, 53, 339-340, (1988)! helix 3 extends from amino-acid 43 to amino-acid 58 of the homeobox peptide. Within the context of the present invention "helix 3" also refers to peptides which may slightly differ in their sequence from the helix 3 of naturally occurring homeodomains, provided that said differences have no influence on the structure of said helix 3 and to its ability to bind to the DNA double helix.
Such polypeptides may for instance result from the substitution of an amino-acid by another, according to the known "groups of substitution" of aminoacids, since it is known in the art that conservative substitution of one hydrophobic, aromatic, aliphatic, acidic or basic residue for another in a peptide frequently does not alter the structural characteristics of said peptide.
Although numerous data, obtained in vitro and in an acellular system, are now available on the homeobox peptide/DNA binding, the effects on cellular functions were unknown up until now. It was not even known whether the homeobox peptides were capable of having an inherent activity or whether their role was simply limited to allowing the homeoprotein/DNA binding.
However, by studying the action of synthetic homeobox peptides on cell cultures, the Inventors have discovered unexpected properties of the said peptides, properties which had never been thought of up until then.
Indeed, they observed that the synthetic homeobox peptides, when they are added to cultured nerve cells, penetrate into all the cells of the culture and that the entry of the peptides into the neurons is followed by accumulation in the nucleus. This accumulation is blocked not only by preincubation with an oligonucleotide containing the consensus sequence ATTA, but also by preincubation with fragments of double-stranded DNA not containing the consensus sequence.
By analysing this penetration process, the Inventors demonstrated the importance therein of the region corresponding to the helix 3+4 (last 27 amino acids of the homeobox peptide).
They also observed the penetration of polypeptides comprising this homeobox peptide.
They also observed this phenomenon, although to a lesser degree, in cultures of cells other than nerve cells.
The Inventors also showed that the accumulation of homeobox peptides in the nucleus was accompanied by intense cell growth and differentiation.
These properties of the homeobox peptides, which have been demonstrated by the Inventors, allow their use for the production of new medicinal products as well as their use in vitro as an agent which is active on cell cultures.
Indeed, it stems from the research work by the Inventors that the homeobox peptides or fragments thereof, are capable of providing new, in particular neurotropic, growth factors and/or new vectors for the transmembrane and intracellular transport of molecules, in particular peptides and oligonucleotides, which are active on the cellular functions.
However, both of these applications correspond to current needs. They have been the subject of various research work of which a brief overview is given below.
The importance of the intracellular vectors for the transport of peptides or oligonucleotides became apparent following the demonstration that some peptides and oligonucleotides, by binding specifically to certain DNA regions, were capable of acting on the cellular functions (for example, proteins activating or repressing the expression of a gene, antisense oligonucleotides and the like).
However, an effective exploitation, in particular for a therapeutic objective, of the properties of these molecules involves delivering them to the site by causing them to cross numerous barriers, in particular the cytoplasmic membrane, separating the extracellular medium from the DNA. However, very often, these molecules, by virtue of their charge and their high molecular mass, are unable to cross this barrier by themselves. Various solutions have been proposed to this problem; some of them, relating to oligonucleotide sequences, are for example mentioned in the introduction in the publication by LEMAITRE et al. Proc. Natl. Acad. Sci. USA 84, 648-652 (1987)!. For their part, these authors propose an approach consisting of covalently binding an oligonucleotide sequence which is complementary to an RNA sequence of the vesicular stomatitis virus (VSV), to (L-lysine) polymer.
The conjugate obtained penetrates into the cells and inhibits specifically the synthesis of the VSV proteins in the infected cells.
These results show the importance of the association between an active macromolecule and a vector for the transport of the said macromolecule. It is therefore particularly desirable to search for new vectors.
In the case of the growth factors which are active on the survival and the differentiation of the neurons, only a small number are currently known; the first one to be identified is the Nerve Growth Factor (NGF). The action of the NGF is exerted essentially on the sensory neurons and the neurons of the sympathetic nervous system; an action on certain cells of the central nervous system and of the immune system has also been detected. The neurotropic activity of the NGF is carried by a subunit (.beta. subunit) of 118 amino acids.
Other substances with neurotropic action have also been described: they are for example, the Ciliary Neurotropic Factor (CNTF) LIN et al, SCIENCE, 246, 1023-1026, (1989); STOCKLI et al. NATURE, 342, 920-923, (1989)!, the Brain Derived Neurotropic Factor (BDNF) LEIBROCK et al., NATURE, 341, 149-152, (1989)!, the Glial Derived Nexin (GDN), a component of the extracellular matrix GLOOR et al., CELL, 47, 687-693, (1986)!,.
More ubiquitous growth factors such as Fibroblast Growth Factor (FGF) PARK and HOLLENBERG, DEV. BIOL., 134, 201-205, (1989)! or the Epidermal Growth Factor (EGF) MORRISON et al., SCIENCE, 238, 72-74, (1987)! also have a neurotropic action.
The mechanism of action of these factors is currently not well known. It has been shown that the NGF penetrates into the neurons via a specific receptor which is a phosphorylated glycoprotein CHAO et al., Science, 232, 518 (1986)!. Inside the nerve cell, the NGF stimulates the synthesis of RNA via a second messenger.
Many experiments show the potential therapeutic importance of neurotropic growth factors.
The use of NGF has for example been suggested in Alzheimer's disease. It has indeed been shown that NGF makes it possible to increase the choline acetyltransferase activity of the cholinergic neurons and to prevent their degeneration MOBLEY et al, Science, 229, 284 (1984)!, KROMER, Science, 235, 214, (1987)!. However, it is known that Alzheimer's disease is associated with degeneration of the cholinergic neurons and with a decrease in the choline acetyltransferase activity.
Experiments carried out in adult rats in which the cholinergic route linking the hippocampus and the septum had previously been destroyed (which results in degeneration of the septal neurons) have shown that the intraventricular injection of NGF allows the survival of the septal neurons as well as the restoration of a normal choline acetyltransferase activity WILL et HEFTI, Behav. Brain. Res., 17,17 (1985)!. The use of neurotropic growth factors has also been envisaged in the case of Parkinson's disease which is linked to a degeneration of the dopaminergic neurons.
Another approach to the treatment of diseases associated with a neuronal degeneration has recently been proposed and appears set, in a new future, for a major development; it is the intracerebral transplantations of cells which are capable of making up for the defective neuronal functions; the use of fetal neurons LINDVALL et al., SCIENCE, 247, 574-577, (1990)! or of transformed cell lines HORELLOU et al., EUR. J. NEUROSCI., 2, 116-119, (1990)! has thus been suggested.
Recently, transformed cells, producing a recombinant NGF, were implanted in the brain of rats at the same time as cholinergic neurons of fetal origin. It was observed that, under these conditions, the survival of the transplanted neurons as well as the neogenesis of the nerve fibres was greatly increased ERNFORS et al., Proc. Natl. Acad. Sci. USA, 86, 4756, (1989)!.
These research studies therefore show that neurotropic growth factors can find particularly advantageous applications in the treatment of disorders resulting from neuronal lesions or degeneration.
A limitation in the use of neurotropic growth factors, however, consists of the small number of growth factors currently known, as well as of their relatively narrow specificity of action which is limited to certain types of neurons. Furthermore, most of these growth factors currently cannot be obtained in sufficient amount for a therapeutic use.
It would therefore be particularly desirable to have neurotropic growth factors which do not possess the disadvantages that have just been mentioned.